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EP1341023B1 - Méthode et software d'apprentissage pour microscope de balayage et microscope de balayage - Google Patents

Méthode et software d'apprentissage pour microscope de balayage et microscope de balayage Download PDF

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Publication number
EP1341023B1
EP1341023B1 EP03100351A EP03100351A EP1341023B1 EP 1341023 B1 EP1341023 B1 EP 1341023B1 EP 03100351 A EP03100351 A EP 03100351A EP 03100351 A EP03100351 A EP 03100351A EP 1341023 B1 EP1341023 B1 EP 1341023B1
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Prior art keywords
user
computer system
spectral
channel
scanning microscope
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EP03100351A
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EP1341023A3 (fr
EP1341023A2 (fr
Inventor
Frank Olschewski
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Leica Microsystems CMS GmbH
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Leica Microsystems CMS GmbH
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes

Definitions

  • the invention relates to a method for user training for a scanning microscope.
  • the invention relates to a scanning microscope with means for receiving a complete spectral scan of an object, wherein the means for recording is provided with spectral selection means, and a computer system having a memory for storing the complete spectral scan in the memory of the computer system.
  • the invention relates to software for user training for a scanning microscope.
  • Microscopes in particular scanning microscopes, consume for training purposes samples that are no longer useful for further investigations due to the radiation exposure (such as bleaching, thermal destruction, etc.).
  • the optimal setting (parameter setting) of a scanning microscope is often tedious for an untrained user. Choosing the wrong parameters can quickly destroy a sample or render it unusable. Since it requires a great deal of time and money to prepare the samples for the microscopic examination, the disadvantage of the previous systems is that samples are likewise consumed in user training without there being any test results or data.
  • the parameters include, for example, the intensity of the individual laser lines irradiated onto the sample, and also the areas of a recorded spectrum to be used for the evaluation and image generation.
  • AOBS Acousto Beam Beam Splitter
  • appropriate parameters of the AOBS must also be set for wavelength selection.
  • German patent application DE 199 44 355.6 discloses an optical arrangement in the beam path of a laser scanning microscope.
  • a spectrally selective element is provided which couples excitation light of the light source of at least one wavelength into the microscope. The scattered on an object and reflected excitation light from the detection beam path is hidden and not coming from the object detection light.
  • the spectrally selective element may be an AOTF (acousto-optical tunable filter), an AOM (acousto-optic modulator) or an AOBS (acousto-optical-beam splitter).
  • the German patent application DE 100 06 800.6 discloses a device for selecting and detecting at least one spectral range of a spectrally fanned out light beam (SP module).
  • SP module spectrally fanned out light beam
  • selection means are provided, which are designed as slides, in order thus to direct parts of the fanned-out light beam to different detectors.
  • the signals from the detectors are then used for imaging.
  • the quality of the picture depends especially on the position of the slider. For an inexperienced user, it is time consuming to find and set the best location.
  • German patent application DE 100 57 948 A1 discloses a method and a device for user guidance in scanning microscopy.
  • the main quality parameters are visualized online, so that a user can adjust the microscope interactively.
  • the user is shown the determined image parameters and he can change selected parameters to improve the Büding or data recording
  • the invention has for its object to provide a method by which a user can learn the settings of a scanning microscope without consuming sample and resources.
  • the objective object is achieved by a method having the features of the characterizing part of claim 1.
  • a further object of the invention is to provide a scanning microscope with which an improvement in the training and training possibilities of a spectral confocal microscope including a cost savings can be achieved.
  • a further object of the invention is to provide a software with which it is possible to carry out the user training for a scanning microscope with a virtual scanning microscope (i.e., no purely software-based learning of the settings).
  • the invention has the advantage that you can play around after a single dose of radiation of a sample with the characteristics of the sample and learn without causing further destruction. Also a demo version for this is possible, which reads the record from a memory (hard disk, RAM, CD-ROM). This considerably minimizes the training time on a confocal scanning microscope and spares valuable samples.
  • Samples showing significant autofluorescence, or mutants can also be started with the lambda scan to experiment with the best conditions for further experimentation. This has a high customer benefit.
  • the values can be loaded directly into the SP module in order to make optimally beautiful images with this data set.
  • the mode of operation of an SP module is adjusted mathematically / simulatively relatively exactly to the mode of operation of the real SP module. Through simulation, the working methods can be transferred to the software level. There, however, you automatically become a kind of inverse filter or something similar (depends a bit on mathematical nomenclature). The true value of the software module comes into play when the AOBS is integrated in the design of the scanning microscope
  • Fig. 1 the embodiment of a confocal scanning microscope 100 is shown schematically. However, this should not be construed as limiting the invention. It is sufficiently clear to the person skilled in the art that the invention can also be implemented with a conventional scanning microscope.
  • the illumination light beam 3 coming from at least one illumination system 1 is directed by a beam splitter or a suitable deflection means 5 to a scan module 7. Before the illumination light beam 3 strikes the deflection means 5, it passes through an illumination pinhole 6.
  • the scan module 7 comprises a gimbal-mounted scanning mirror 9, which guides the illumination light beam 3 through a scan optics 12 and a microscope optics 13 over or through an object 15. The illumination light beam 3 is guided over the object surface with non-transparent objects 15.
  • the illumination light beam 3 can also be guided through the object 15.
  • non-luminous preparations are optionally prepared with a suitable dye (not shown, since established prior art). This means that different focal planes of the object are sequentially scanned by the illumination light beam 3.
  • a position sensor 11 With the scan module 7, a position sensor 11 is connected, which determines the position data of the recorded image data. The subsequent composition of position data and the image data then yields a two- or three-dimensional frame (or image) of the object 15.
  • the illumination light beam 3 coming from the illumination system 1 is shown as a solid line.
  • the light emanating from the object 15 defines a detection light beam 17.
  • the detection light beam 17 originating or defined by the object 15 is shown in FIG. 1 as a dashed line. In the detector 19 electrical, to the power of the light emitted from the object 15, proportional detection signals are generated.
  • the computer system 23 is assigned at least one peripheral device 27.
  • the peripheral device may be, for example, a display on which the user receives instructions for setting the scan microscope or can take the current setup and also the image data in graphic form.
  • a user interface is also shown on the display, as shown in FIG. 4, for example.
  • an input means is associated with the computer system 23, which consists for example of a keyboard 28, a setting device 29 for the components of the microscope system and a mouse 30.
  • FIG. 2 shows the embodiment of a scanning microscope in which an SP module 20 is used as selection means in front of the at least one detector 19 is arranged. All other elements shown in Fig. 2 are identical to those of Fig. 1, so that they need not be mentioned again in the description of FIG.
  • the SP module 20 (FIG. 2) records a complete lambda scan, ie for each object point all wavelengths emanating from the object 15 are recorded.
  • the data is transmitted to the computer system 23 and can then be displayed on the display 27 in a user-definable manner.
  • the detection light beam 17 is spatially spectrally split by a prism 31. Another possibility of spectral splitting is the use of a reflection or transmission grating.
  • the spectrally split light fan 32 is focused with the focusing optics 33 and then strikes a mirror shutter assembly 34, 35.
  • the mirror shutter assembly 34, 35, the means for spectral spatial splitting (prism 31), the focusing optics 33 and the detectors 36 and 37 are combined referred to as SP module 20 (or Mutibanddetektor).
  • SP module 20 or Mutibanddetektor
  • a desired part of the spectrum can be selected by means of the mirror shutter arrangement 34, 35.
  • the user slides on the user interface, which effect an adjustment of the mirror shutter arrangement 34, 35 corresponding to the selection in the SP module 20.
  • the mirror shutter assemblies 34, 35 are displaceable in the directions illustrated by the double arrows so that the spectral detection ranges of the light supplied to the detectors 36, 37 are continuously adjustable. It is possible, however, not shown for the sake of clarity, to install even more detectors and assign more mirrors.
  • the detectors 36, 37 electrical, proportional to the output of the object 15 outgoing detection light beam 17 of the respective spectral range detection signals are generated, which in the computer system 23 to be assigned to the detected in the beam deflection device by means of a position sensor position signals.
  • FIG. 3 shows the basic structure for user training for a scanning microscope 100.
  • This user training allows a user to learn the setting operations of a scanning microscope 100 without requiring an object 15 during the entire learning phase.
  • a software module 102 Connected to the scanning microscope 100 is a software module 102 that is designed to be interactive.
  • the operation of the SP module 20 (FIG. 2) is simulated, that is, spectral bands are separated from the data set, accumulated, grouped into channels, displayed in multiple colors. All possibilities of the software of a confocal scanning microscope come into consideration. In principle, the software looks like a user interface (see Fig.4).
  • I i ⁇ ⁇ min + i ⁇ ⁇ ⁇ min + ( i + 1 ) ⁇ ⁇ I ( ⁇ )
  • the scan is determined by the system design or the parameter setting.
  • the dimensionality n is freely adjustable.
  • the data supplied by the scanning microscope 100 or a scan detected with the scanning microscope is held in the RAM of the computer system 23.
  • the computer system 23 switches to a simulation mode of the SP module 30.
  • a simulator 104 is connected to the software module 102, which shows the user an exact user interface with the standard components of the SP module operating software.
  • FIG. 4a shows part of a schematic embodiment of a user interface 40 with which the user can define a number of desired channels.
  • 40 selection slides 41 green , 41 red , 41 blue or 41 gray are shown on the user interface, with which the user can tune the various spectral bands.
  • the user adjusts a spectral band, and as a result, the mirror slides 34 and 35 are moved in the SP module 30. The result is that optically the spectral band is separated and displayed.
  • the simulation is achieved by the computer system 23 simulating this optical separation by a vector for each desired channel I ⁇ channel with the dimensionality of the above vector I ⁇ is generated by setting the values for I i which are in the selected band to one.
  • the computer system 23 calculates a pixel by pixel image for each desired channel through the linear combination (see Equation 2).
  • I channel 1 ⁇ I ⁇ channel ⁇ ⁇ I ⁇ .
  • the calculated data of the channels are displayed to the user on the display 27, in which representation all possible display modes (overlay, volume rendering, etc.) can be included.
  • the simulator 104 is connected to the computer system 23.
  • the user can, without burdening the object 15 (by radiation, thermal, etc.), continue to do so until he is satisfied with the image displayed on the display 27.
  • the setting is saved and made available as a filter macro or setting macro of the SP module 30 for further work with the same object 15 or similar objects.
  • the scanning microscope is also shown schematically, and the user is provided with a large number of setting options.
  • a first laser 45 and a second laser 47 are provided, both of which are schematically illustrated as a box.
  • the first laser 45 is, for example, an argon UV laser (ArUV laser) which emits light of a first wavelength of 351 nm and light of a second wavelength of 364 nm.
  • the second laser 45 is, for example, an argon krypton laser (ArKr laser) of the light of a first wavelength of 476 nm, light of a second wavelength of 514 nm, light of a third wavelength of 568 nm and light of a fourth wavelength of 647nm emitted.
  • a slider 50 is provided for each of the available wavelengths, via which the percentage of the laser power of the respective wavelength is adjustable.
  • a display 46, 48 is provided in each box, which indicates the operating state of the respective laser, or via which the laser is switched on or off.
  • a data structure 52 is shown, as the data are stored on the memory of the computer system 23.
  • the display also shows the sample 54 and a light beam 55 coming from the lasers 45, 47, a light beam 56 transmitted by the sample 54, and a light beam 57 reflected by the sample 54. The light beams are directed by a schematically illustrated beam deflecting device 58.
  • the light beam 57 coming from the sample 54 points to a representation of a spectrum 60.
  • the lines emitted by the first and second lasers 45, 47 are plotted.
  • the intensity and the spectral position of the light 57 reflected by the sample 54 are shown in the spectrum 60.
  • a first intensity curve 62, a second intensity curve 64, and a third intensity curve 66 are shown in the spectrum 60.
  • a scale 68 is provided which serves as an orientation aid for the selection slides 41 green , 41 red , 41 blue or 41 gray arranged underneath. The selection slides 41 green , 41 red , 41 blue or 41 gray are moved with the mouse or a similar means on the user interface 40.
  • a first detector 74, a second detector 75, a third detector 76 and a fourth detector 77 are also shown schematically as a box.
  • a display 78 is provided in each box.
  • the display 78 is configured as a drop-down display, so that the user can easily select a different dye.
  • each box is assigned a color indication 79, which indicates how the signals of the respective detector are used for image generation on the display (see FIG. 4b).
  • the operating state of each detector is indicated in each box via an activatable box 80.
  • the light transmitted by the sample 54 is associated with a fifth detector 82, which also has the display 78 for the dye detected by the detector 82, the color indicator box 79, and the activatable box 80.
  • the images of a real sample or a virtual sample are shown as they arise when the user on the user interface 40, the selection slider 41 green , 41 red , 41 blue or 41 gray changed and thereby other areas of the spectrum selects for image generation.
  • the intensity recorded by the first detector 74 is used to generate a green image 63.
  • the intensity received by the second detector 75 is used to generate a red image 65.
  • the intensity received by the third detector 76 is used to generate a blue image 67. It goes without saying that the images differ in that different or additional structures 63a, 65a and 67a become visible in the respectively selected spectral regions.
  • the intensity I is plotted as a function of the wavelength ⁇ .
  • the spectrum 90 shown in FIG. 5 can be generated, for example, by means of a lambda scan or retrieved from a database in which spectra 90 are stored for learning purposes.
  • the spectrum 90 can be represented by a vector I ⁇ with the individual components a 1 , a 2 to a n (n-dimensional) are represented. Below the abscissa, the selected regions of the spectrum are identified by first, second and third rectangles 91, 92 and 93. is shown.
  • the simulation is achieved in that the computer system 23 simulates this optical separation by the vector for the channel or spectral range defined by the second rectangle 92 I ⁇ channel 2 is generated with the dimensionality of the above-mentioned vector I.
  • the values of I i which are within the second rectangle 92 are set to one.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)

Claims (17)

  1. Méthode de formation d'utilisateur pour un microscope à balayage (100), caractérisée par les étapes suivantes :
    a) extraire un balayage spectral complet d'une mémoire du système informatique (23) ;
    b) simuler une sélection spectrale, l'utilisateur définissant plusieurs canaux provenant du balayage spectral complet ;
    c) ajuster des moyens de sélection spectrale sur le système réel, le système informatique (23) simulant la séparation optique ;
    d) générer et représenter une image pour chaque canal défini par l'utilisateur ; et
    e) répéter les étapes c) à e) ci-dessus jusqu'à ce que les images générées correspondent au contenu d'information déterminé par l'utilisateur.
  2. Méthode selon la revendication 1, caractérisée en ce que les étapes suivantes .
    a) enregistrer un balayage spectral complet d'un objet (15) ; et
    b) stocker le balayage spectral complet dans une mémoire du système informatique (23).
  3. Méthode selon la revendication 1 ou 2, caractérisée en ce que la simulation est exécutée à l'aide d'un module SP (20), dans laquelle une interface utilisateur exacte (40) avec les composants standard du logiciel d'utilisation pour le module SP (20) est affichée à destination de l'utilisateur sur un affichage (27) affecté au système informatique (23).
  4. Méthode selon la revendication 1, caractérisée en ce que lors de l'enregistrement d'un balayage complet, un vecteur d'intensité spectral pleinement valable I ‾
    Figure imgb0026
     est enregistré pour chaque pixel, dans laquelle I ‾ = ( I 1 ⋮ I n ) ,   I i = ∫ λ min + i Δ λ λ min + ( i + 1 ) Δ λ I ( λ )
    Figure imgb0027
    correspond au balayage spectral complet par le module SP (20) et Δλ est ajustable librement sur le module SP (20).
  5. Méthode selon la revendication 1, caractérisée en ce que les moyens de sélection interactifs, représentés sur l'interface utilisateur (40), sont réalisés comme des glissières de miroir (34, 35) permettant de séparer et d'afficher une bande de façon optique.
  6. Méthode selon la revendication 5, caractérisée en ce que le système informatique (23) simule une séparation optique en ce que pour chaque canal souhaité un vecteur IKanal avec la dimensionnalité du vecteur d'intensité spectral pleinement valable I est généré, dans laquelle les composantes Ii du vecteur d'intensité spectral IKanal qui se trouvent dans la bande séparée sont réglées sur un.
  7. Méthode selon la revendication 6, caractérisée en ce que le système informatique (23) calcule pour chaque canal souhaité pixel par pixel une image par l'intermédiaire de la combinaison linéaire I Kanal = 1 ‖ I ‾ Kanal ‖ 〈 I ‾ , I ‾ Kanal 〉
    Figure imgb0028
  8. Méthode selon la revendication 1, caractérisée en ce que plusieurs modes de représentation sont impliqués dans la représentation.
  9. Méthode selon la revendication 1, caractérisée en ce que pendant la formation d'utilisateur interactive, les réglages simulés sont repris comme un enregistrement de configuration dans la commande de microscope.
  10. Microscope à balayage (100), comprenant des moyens pour enregistrer un balayage spectral complet d'un objet (15), dans lequel les moyens d'enregistrement sont munis de moyens de sélection spectrale, d'un système informatique (23) avec une mémoire pour le stockage du balayage spectral complet dans la mémoire du système informatique (23), caractérisé en ce qu'un module de simulateur (104) est prévu qui reproduit pour l'utilisateur d'après la sélection de celui-ci de certains canaux provenant du balayage spectral complet une sélection spectrale sur un affichage (27) affecté au système informatique (23).
  11. Microscope à balayage (100) selon la revendication 10, caractérisé en ce que le module de simulateur (104) comprend un logiciel de simulation qui génère avec l'utilisateur un réglage optimal des moyens de sélection à partir des différentes sélections spectrales représentées sur l'affichage (27).
  12. Microscope à balayage selon la revendication 10, caractérisé en ce que les moyens d'enregistrement d'un balayage complet sont réalisés comme un module SP (20) et que les moyens de sélection spectrale sont réalisés comme des glissières de miroir (34, 35) permettant de séparer une bande de façon optique.
  13. Microscope à balayage (100) selon la revendication 10, caractérisé en ce que le système informatique (23) calcule pour chaque canal souhaité pixel par pixel une image par l'intermédiaire de la combinaison linéaire I Kanal = 1 ‖ I ‾ Kanal ‖ 〈 I ‾ , I ‾ Kanal 〉
    Figure imgb0029
    dans lequel, pour chaque canal souhaité, un vecteur I ‾ Kanal
    Figure imgb0030
     avec la dimensionnalité du vecteur d'intensité spectral pleinement valable I est généré, dans lequel les composantes Ii du vecteur d'intensité spectral I ‾ Kanal
    Figure imgb0031
     qui se trouvent dans la bande séparée sont réglées sur un.
  14. Microscope à balayage (100) selon la revendication 13, caractérisé en ce que plusieurs modes de représentation sont accessibles pour la représentation.
  15. Microscope à balayage (100) selon la revendication 10, caractérisé en ce que le module de simulateur (104) comprend une interface utilisateur (40), dans lequel l'interface utilisateur (40) affiche pour l'utilisateur l'image de l'objet enregistré dans différents modes de représentation et reproduit schématiquement des moyens réglables du système de microscope à balayage (100), dans lequel les moyens réglables peuvent être ajustés par des moyens d'entrée (28, 29, 30) du système informatique (23).
  16. Logiciel sur un support de données, caractérisé en ce que le logiciel exécute sur un système informatique (23) usuel une formation d'utilisateur pour un microscope à balayage (100), dans lequel la formation d'utilisateur est caractérisée par les étapes suivantes :
    • extraire un balayage spectral complet d'une mémoire du système informatique (23) ;
    • simuler une sélection spectrale, l'utilisateur définissant plusieurs canaux provenant du balayage spectral complet ;
    • ajuster des moyens de sélection spectrale sur le système réel, le système informatique (23) simulant la séparation optique ;
    • générer et représenter une image pour chaque canal défini par l'utilisateur ; et
    • répéter les étapes c) à e) ci-dessus jusqu'à ce que les images générées correspondent au contenu d'information déterminé par l'utilisateur.
  17. Logiciel selon la revendication 16, caractérisé en ce que le système informatique (23) se présente sous la forme d'un PC usuel, d'un assistant numérique personnel usuel, d'un téléphone usuel ou d'un système domestique multimédia (hi-fi, vidéo) usuel ou d'une console Gameboy usuelle.
EP03100351A 2002-02-20 2003-02-17 Méthode et software d'apprentissage pour microscope de balayage et microscope de balayage Expired - Lifetime EP1341023B1 (fr)

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DE10206979A DE10206979A1 (de) 2002-02-20 2002-02-20 Verfahren zum Benutzertraining für ein Scanmikroskop, Scanmikroskop und Software zum Benutzertraining für ein Scanmikroskop
DE10206979 2002-02-20

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EP1341023A3 (fr) 2003-11-19
DE50304624D1 (de) 2006-09-28
US7218762B2 (en) 2007-05-15
DE10206979A1 (de) 2003-08-21
EP1341023A2 (fr) 2003-09-03

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